1- Making Density Gradients

# 1- Making Density Gradients

 Views: 193   |  Added: 20-02-2012 Rate Presentation:
Description:
Discontinuous gradient by overlayering. Discontinuous gradient by underlayering. . Diffusion of a discontinuous gradient. Rapid formation of continuous from discontinuous gradient. Two-chamber device for continuous gradients. The Gradient Master. B. . Gradient Master profiles from 10% and 40% iodixanol at 80
1- Making Density Gradients

An Image/Link below is provided (as is) to

Download Policy: Content on the Website is provided to you AS IS for your information and personal use only and may not be sold or licensed nor shared on other sites. SlideServe reserves the right to change this policy at anytime. While downloading, If for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

1. 1- Making Density Gradients Pre-formed discontinuous gradients Pre-formed continuous gradients Self-generated gradients Training File 2 topicsTraining File 2 topics

2. Discontinuous gradient by overlayering If we make up, for example 3 aqueous solutions of, for example, iodixanol (10%, 20% and 30%, w/v) we can make a discontinuous gradient by overlayering; each solution (starting with the densest) is overlayered with successively lighter solutions from either a pipette or syringe attached to a wide-bore flat-tipped metal tube (cannula). Overlayering is best accomplished if the tube is tilted (as in C) and the tip of the pipette or syringe placed approx 0.5 cm above the meniscus.If we make up, for example 3 aqueous solutions of, for example, iodixanol (10%, 20% and 30%, w/v) we can make a discontinuous gradient by overlayering; each solution (starting with the densest) is overlayered with successively lighter solutions from either a pipette or syringe attached to a wide-bore flat-tipped metal tube (cannula). Overlayering is best accomplished if the tube is tilted (as in C) and the tip of the pipette or syringe placed approx 0.5 cm above the meniscus.

3. Discontinuous gradient by underlayering The less commonly used method of underlayering (low density end first) is in fact the more satisfactory method. It is only easily accomplished using a syringe and metal cannula and the hemispherical section of the bottom of the tube aids smooth flow. After adding the least dense solution, the next most dense solution is taken into the syringe – if for example you need to layer 3 ml, then take up approx 5 ml and expel the liquid to the 4 ml mark (this ensures there are no air bubbles in the cannula. Wipe the outside of the cannula with a tissue to remove any excess liquid and introduce the tip of the cannula to the bottom of the tube, moving it smoothly down the wall of the tube. Smoothly introduce 3 ml of the solution. Depressing the syringe plunger to the 1ml mark rather than to the bottom of the barrel is both more accurate and ensures any air bubbles, trapped on the plunger, do not enter the tube. After a few seconds withdraw the syringe, again keeping the tip of the cannula against the wall of the tube and repeat the procedure. The less commonly used method of underlayering (low density end first) is in fact the more satisfactory method. It is only easily accomplished using a syringe and metal cannula and the hemispherical section of the bottom of the tube aids smooth flow. After adding the least dense solution, the next most dense solution is taken into the syringe – if for example you need to layer 3 ml, then take up approx 5 ml and expel the liquid to the 4 ml mark (this ensures there are no air bubbles in the cannula. Wipe the outside of the cannula with a tissue to remove any excess liquid and introduce the tip of the cannula to the bottom of the tube, moving it smoothly down the wall of the tube. Smoothly introduce 3 ml of the solution. Depressing the syringe plunger to the 1ml mark rather than to the bottom of the barrel is both more accurate and ensures any air bubbles, trapped on the plunger, do not enter the tube. After a few seconds withdraw the syringe, again keeping the tip of the cannula against the wall of the tube and repeat the procedure.

5. Rapid formation of continuous from discontinuous gradient If the tube containing the discontinuous gradient is capped and very carefully and smoothly rotated to a horizontal position, the interfacial area increases and the distance between each interface decreases, so diffusion and formation of a continuous gradient will occur much more rapidly. At room temperature, a continuous gradient of iodixanol in a 14 ml tube can be formed in approx 45 min. This should not be attempted the morning after the night before.If the tube containing the discontinuous gradient is capped and very carefully and smoothly rotated to a horizontal position, the interfacial area increases and the distance between each interface decreases, so diffusion and formation of a continuous gradient will occur much more rapidly. At room temperature, a continuous gradient of iodixanol in a 14 ml tube can be formed in approx 45 min. This should not be attempted the morning after the night before.

6. Two-chamber device for continuous gradients Two identical chambers (A and B) connected by a tapped channel (T) contain equal volumes of the high density and low density solutions respectively and identical magnetic stirring bars (SB). Chamber B, which sits on a magnetic stirrer (M), bears a delivery tube which reaches to the bottom of a centrifuge tube via a low-pulsation peristaltic pump (P). When the levels of liquid in the two chambers fall synchronously, dense solution from A mixes continually with the lighter solution in B so that the pump delivers a solution of ever-increasing density to the bottom of the centrifuge tube. The two stirring bars ensure that the level of liquid in A and B is the same. If the two solutions have very different densities and if the stirring is not sufficiently vigorous, the dense solution may flow under the lighter solution. If the placement of the two solutions is reversed, then the tendency of the less dense solution two float up through the denser solution in B will improve mixing considerably. In this format the tip of the delivery tube needs to be placed against the wall of the tube, near its top, so that solution of decreasing density flows to the bottom. If the flow down the wall of the tube is not continuous (but in the form of drops) this can also cause mixing. Use of the Labconco Auto Densi-flow machine (see Graphic 31) to deliver the gradient into the tube avoids this problem totally in the dense-end first mode. A single two-chamber gradient maker can be used to create multiple gradients but any manifold in the delivery line must be situated before P.Two identical chambers (A and B) connected by a tapped channel (T) contain equal volumes of the high density and low density solutions respectively and identical magnetic stirring bars (SB). Chamber B, which sits on a magnetic stirrer (M), bears a delivery tube which reaches to the bottom of a centrifuge tube via a low-pulsation peristaltic pump (P). When the levels of liquid in the two chambers fall synchronously, dense solution from A mixes continually with the lighter solution in B so that the pump delivers a solution of ever-increasing density to the bottom of the centrifuge tube. The two stirring bars ensure that the level of liquid in A and B is the same. If the two solutions have very different densities and if the stirring is not sufficiently vigorous, the dense solution may flow under the lighter solution. If the placement of the two solutions is reversed, then the tendency of the less dense solution two float up through the denser solution in B will improve mixing considerably. In this format the tip of the delivery tube needs to be placed against the wall of the tube, near its top, so that solution of decreasing density flows to the bottom. If the flow down the wall of the tube is not continuous (but in the form of drops) this can also cause mixing. Use of the Labconco Auto Densi-flow machine (see Graphic 31) to deliver the gradient into the tube avoids this problem totally in the dense-end first mode. A single two-chamber gradient maker can be used to create multiple gradients but any manifold in the delivery line must be situated before P.

8. Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time Increasing the time of rotation generally makes the gradient more linear and more shallowIncreasing the time of rotation generally makes the gradient more linear and more shallow

9. Swinging-bucket rotor There are two traditional types of rotor: swinging-bucket and fixed-angle. The swinging-bucket rotor is by far the most widely used for density gradient centrifugation. In the swinging-bucket rotor, at rest, the tube and bucket are vertical and the meniscus of the liquid is at 90° to the earth’s vertical centrifugal field. During acceleration of the rotor the bucket, tube and meniscus reorient through 90° in the spinning rotor’s radial centrifugal field. Any gradient in the tube reorients with the tube so that interfaces are also always perpendicular to the centrifugal field.There are two traditional types of rotor: swinging-bucket and fixed-angle. The swinging-bucket rotor is by far the most widely used for density gradient centrifugation. In the swinging-bucket rotor, at rest, the tube and bucket are vertical and the meniscus of the liquid is at 90° to the earth’s vertical centrifugal field. During acceleration of the rotor the bucket, tube and meniscus reorient through 90° in the spinning rotor’s radial centrifugal field. Any gradient in the tube reorients with the tube so that interfaces are also always perpendicular to the centrifugal field.

11. Sedimentation path length of rotors In a swinging-bucket rotor the sedimentation path length is the length of the tube. In a vertical rotor the sedimentation path length is the diameter of the tube. The vertical rotor is therefore the most efficient rotor; at the same RCF, particles will reach their banding density much more quickly in a vertical rotor than in a swinging-bucket rotor. The fixed-angle rotor would occupy an intermediate position. But in a vertical rotor sedimenting particles cannot encounter the wall of the tube in the same way as they do in a fixed-angle rotor. In addition, for tubes of the same volume and dimensions (rotating at the same rpm), the hydrostatic pressure experienced by a particle is much less in a vertical rotor than in a swinging-bucket rotor, since the height of the liquid column is much smaller. The hydrostatic pressure is a function of the square of the height of the liquid column and this pressure has been shown to damage some organelles. In short, for density gradient centrifugation, the vertical rotor has none of the disadvantages of either the swinging-bucket rotor or the fixed-angle rotor.In a swinging-bucket rotor the sedimentation path length is the length of the tube. In a vertical rotor the sedimentation path length is the diameter of the tube. The vertical rotor is therefore the most efficient rotor; at the same RCF, particles will reach their banding density much more quickly in a vertical rotor than in a swinging-bucket rotor. The fixed-angle rotor would occupy an intermediate position. But in a vertical rotor sedimenting particles cannot encounter the wall of the tube in the same way as they do in a fixed-angle rotor. In addition, for tubes of the same volume and dimensions (rotating at the same rpm), the hydrostatic pressure experienced by a particle is much less in a vertical rotor than in a swinging-bucket rotor, since the height of the liquid column is much smaller. The hydrostatic pressure is a function of the square of the height of the liquid column and this pressure has been shown to damage some organelles. In short, for density gradient centrifugation, the vertical rotor has none of the disadvantages of either the swinging-bucket rotor or the fixed-angle rotor.

15. Near-vertical rotors are best of all A problem with a vertical rotor is that it is necessary to create a gradient whose (a) highest density is greater than that of the densest particle in the sample and (b) lowest density is less than that of the least dense particle. If this is not the case then the most and least dense particles will reach the wall of the rotor. During the subsequent reorientation of the gradient and during the harvesting of the gradient, this material may contaminate the rest of the gradient. In a near vertical rotor, if this situation occurs, dense pelleted material or light floating material will not create such problems. A problem with a vertical rotor is that it is necessary to create a gradient whose (a) highest density is greater than that of the densest particle in the sample and (b) lowest density is less than that of the least dense particle. If this is not the case then the most and least dense particles will reach the wall of the rotor. During the subsequent reorientation of the gradient and during the harvesting of the gradient, this material may contaminate the rest of the gradient. In a near vertical rotor, if this situation occurs, dense pelleted material or light floating material will not create such problems.

16. Iodixanol self-generated gradient requirements Rotors with a short sedimentation path length ~ 17 mm RCF of 180,000-350,000gav For rapid, efficient formation of all self-generated gradients, a rotor with a relatively short sedimentation path length is required. Only in these rotors can gradients that are more or less linear with volume be created in a reasonably short time < 4h). So vertical rotors are the rotors of choice, although some small volume fixed-angle rotors also fit the bill. Long path length rotors, such as swinging-bucket rotors tend to produce gradients which retain the very shallow middle section even after long periods of centrifugation. Although self-generated gradients will form at RCFs as low as 180,000gav, the optimal formation occurs at approx 350,000gav.For rapid, efficient formation of all self-generated gradients, a rotor with a relatively short sedimentation path length is required. Only in these rotors can gradients that are more or less linear with volume be created in a reasonably short time < 4h). So vertical rotors are the rotors of choice, although some small volume fixed-angle rotors also fit the bill. Long path length rotors, such as swinging-bucket rotors tend to produce gradients which retain the very shallow middle section even after long periods of centrifugation. Although self-generated gradients will form at RCFs as low as 180,000gav, the optimal formation occurs at approx 350,000gav.

18. Effect of RCF and [iodixanol] in Beckman VTi65.1 (3 h) The progression of the shape of the gradient from S-shaped to more linear is also seen by keeping the time constant and increasing the RCF. Tubes for the Beckman VTi65.1 rotor filled with either 15% or 30% iodixanol were centrifuged for 3 h at either 170,000gav or 353,000gav; then unloaded from the top (see Slides 30-32) into 11 fractions whose density was determined by refractive index. Another factor which influences the shape of the gradient is temperature.The progression of the shape of the gradient from S-shaped to more linear is also seen by keeping the time constant and increasing the RCF. Tubes for the Beckman VTi65.1 rotor filled with either 15% or 30% iodixanol were centrifuged for 3 h at either 170,000gav or 353,000gav; then unloaded from the top (see Slides 30-32) into 11 fractions whose density was determined by refractive index. Another factor which influences the shape of the gradient is temperature.

19. Removal of banded material using a syringe Obvious bands may be harvested from an open-topped tube using a syringeObvious bands may be harvested from an open-topped tube using a syringe

20. Collecting a band from a sealed tube Clearly banded material in an open-topped tube can be simply recovered using a syringe and metal cannula. Banded material in a sealed tube may be harvested by first piercing the tube close to the top with a syringe needle attached to an “open” syringe. With the bezel uppermost, a second needle is inserted just below the band to be harvested. When the plunger of the syringe is withdrawn, air can enter the tube to displace the liquid, through the upper syringe.Clearly banded material in an open-topped tube can be simply recovered using a syringe and metal cannula. Banded material in a sealed tube may be harvested by first piercing the tube close to the top with a syringe needle attached to an “open” syringe. With the bezel uppermost, a second needle is inserted just below the band to be harvested. When the plunger of the syringe is withdrawn, air can enter the tube to displace the liquid, through the upper syringe.

22. Upward displacement In upward displacement, a dense solution (Perfluorodecalin – an inert low viscosity fluorocarbon with a density of approx.1.9 g/ml is recommended) is introduced to the bottom of the tube and the gradient harvested low density end first using a simple conical collection device (fashioned from a block of acrylic) on top of the tube. The Perfluordecalin may be introduced from a burette and pump via a tube inserted through the collection head.This method can be used with either thin-walled or thick-walled tubes. The Beckman Fraction Recovery system also includes a device for collection from the top of the tube. Alternatively (for thin-walled tubes only) the Perfluorodecalin may be directed into the bottom of the centrifuge tube via the hollow needle of a tube puncture device. The burette allows easy collection of equal volume fractions and this is the only guaranteed and accurate way of accomplishing this task.In upward displacement, a dense solution (Perfluorodecalin – an inert low viscosity fluorocarbon with a density of approx.1.9 g/ml is recommended) is introduced to the bottom of the tube and the gradient harvested low density end first using a simple conical collection device (fashioned from a block of acrylic) on top of the tube. The Perfluordecalin may be introduced from a burette and pump via a tube inserted through the collection head.This method can be used with either thin-walled or thick-walled tubes. The Beckman Fraction Recovery system also includes a device for collection from the top of the tube. Alternatively (for thin-walled tubes only) the Perfluorodecalin may be directed into the bottom of the centrifuge tube via the hollow needle of a tube puncture device. The burette allows easy collection of equal volume fractions and this is the only guaranteed and accurate way of accomplishing this task.

24. Labconco Auto Densi-flow linked to a Gilson FC205 Fraction Collector The ultimate sophistication in gradient collection – a Labconco Auto Dens-flow linked to a Gilson Fraction Collector; the gradient from a 38 ml tube being collected in a Greiner Bio-One Master Block. The Auto-Densi-Flow has been used for Beckman Optiseal tubes as small as 3 ml.The ultimate sophistication in gradient collection – a Labconco Auto Dens-flow linked to a Gilson Fraction Collector; the gradient from a 38 ml tube being collected in a Greiner Bio-One Master Block. The Auto-Densi-Flow has been used for Beckman Optiseal tubes as small as 3 ml.

Presentation Statistics
Views on SlideServe : 193
Views from Embeds : 0

Other Related Presentations